Abstract
An assay for telomerase activity based on asymmetric polymerase chain reaction (A-PCR) on magnetic beads (MBs) and subsequent application of cycling probe technology (CPT) is described. In this assay, the telomerase reaction products are immobilized on MBs, which are then washed to remove PCR inhibitors that are commonly found in clinical samples. The guanine-rich sequences (5'-(TTAGGG)n-3') of the telomerase reaction products are then preferentially amplified by A-PCR, and the amplified products are subsequently detected via CPT, where a probe RNA with a fluorophore at the 5' end and a quencher at the 3' end is hydrolyzed by RNase H in the presence of the target DNA. The catalyst-mediated cleavage of the probe RNA enhances fluorescence from the 5' end of the probe. The assay allowed us to successfully detect HeLa cells selectively over normal human dermal fibroblast (NHDF) cells. Importantly, this selectivity produced identical results with regard to detection of HeLa cells in the absence and presence of excess NHDF cells; therefore, this assay can be used for practical clinical applications. The lower limit of detection for HeLa cells was 50 cells, which is lower than that achieved with a conventional telomeric repeat amplification protocol assay. Our assay also eliminated false-negative results caused by PCR inhibitors. Furthermore, we show that this assay is appropriate for screening among G-quadruplex ligands to find those that inhibit telomerase activity.
Highlights
Telomerase is responsible for the elongation of telomere DNA, which comprises repeated units of guanine rich (G-rich) sequences (e.g., 5'-TTAGGG-3') at chromosome ends [1,2,3]
TS primer (Table 1), which serves as a substrate for telomerase elongation; (ii) The biotin-labelled telomerase reaction products are immobilized on streptavidin-coated magnetic beads (MBs) via interaction between biotin and streptavidin; (iii) MBs coated with telomerase products are washed to remove sample contaminants including polymerase chain reaction (PCR) inhibitors; (iv) The G-rich sequences of the telomerase products are preferentially amplified by asymmetric polymerase chain reaction (A-PCR); (v) Amplified G-rich sequences are detected via cycling probe technology (CPT)
Based on the results described above, telomerase activity in HeLa cells lysate could be detected as follows: (i) Telomerase reaction carried out in the presence of λ DNA at 37 °C for 60 min; (ii) Telomerase reaction products immobilized on MBs at 25 °C for 30 min, and washed with clean buffer; (iii) Telomerase reaction products on MBs amplified over 30 cycles of A-PCR with μM TS primer and 1 μM CX-ext primer; (iv) A-PCR products are detected by CPT with probe 2
Summary
Telomerase is responsible for the elongation of telomere DNA, which comprises repeated units of guanine rich (G-rich) sequences (e.g., 5'-TTAGGG-3') at chromosome ends [1,2,3]. Other groups proposed telomerase assays with novel signal amplification processes involving enzymes [24,25,26,27], DNAzymes [28,29,30], and nanoparticles [31,32,33,34] instead of PCR. Some of these assays with novel amplification reactions detected telomerase activity with high sensitivity, the enzymes used for catalysis-based amplification may be inhibited by components in clinical samples
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
